DESCRIPTION: All cells incorporate Zn(II) into a wide variety of essential enzymes, gene regulatory proteins, vesicles and nucleic acids. While serum levels are in the micromolar range, typical eukaryotic cells concentrate this element to a total intracellular level in the millimolar range. Zinc is clearl an essential element that a cell must accumulate against a thermodynamic gradient. In spite of its importance in regulation of normal growth and development, few of the mechanisms responsible for maintaining a critical intracellular level of this ion are known. A wide variety of studies suggest that zinc deficiency can pre-dispose individuals to a variety of disorders. Furthermore, zinc is a critical component in immune system functions. Deficiency of this element can lead to significant health problems in individuals infected with the retroviral infections. Marginal zinc deficiency in human beings is a common and perhaps significant public health problem; however, even the most basic cellular and molecular mechanisms for zinc uptake or response to zinc deficiency are poorly understood. The principal thrust of the proposed research is to characterize some of the fundamental biochemistry and cell biology of zinc homeostasis machinery in eukaryotic cells. The specific aims focus on characterizing how changes in Zn(II) concentration are modulated by a recently discovered class of vesicles that are involved in Zn(II) uptake, storage and utilization. To achieve these goals, a series of new tools for determining subcellular concentrations of Zn(II) will be developed. Zn-responsive fluorescent sensors are being develope to study changes in zinc distribution in living cells. Like the family of calcium probes, the zinc probes will be useful in studying changes in Zn(II) concentration in specialized cells, such as hippocampal neurons, in real time. Methods from the fields of cell biology, molecular biology, and chemistry will be employed to characterize components of the cellular machinery that maintain essential levels of this element.